Method and apparatus for characterizing and counting particles, in particular, biological particles

ABSTRACT

The present invention relates to a method and an apparatus for the characterization and/or the counting of particles by means of non uniform, time variable force fields and integrated optical or impedance meter sensors. The force fields can be of positive or negative dielectrophoresis, electrophoresis or electro-hydrodynamic motions, characterized by a set of stable equilibrium points for the particles (solid, liquid or gaseous); the same method is suitable for the manipulation of droplets (liquid particles) by exploiting effects known to the international scientific community with the name of Electro-wetting on dielectric. The aim of the present invention is to act on the control of the position of each particle which is present in the sample, for the purpose of displacing such particles in a deterministic or statistical way, in order to detect their presence with the integrated optical or impedance meter sensors and/or characterize their type, for the purpose of counting or manipulating them in an efficient way.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a U.S. National Phase Application under 35 USC §371of International Application PCT/IB2006/000636 filed on 22 Mar. 2006designating the United States, which claims priority to ItalianApplication No. BO2005A000646, filed Oct. 26, 2005. Priority to each ofthe foregoing PCT and Italian national applications is claimed hereinunder all applicable laws and provisions, and each of said priorityapplications is incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to methods and apparatus for thecharacterization and/or the counting of particles. The invention mainlyfinds an application in the implementation of biological protocols onsingle cells.

STATE OF THE ART

The PCT/WO 00/69565 patent application to G. Medoro describes anapparatus and a method for the manipulation and the detection ofparticles through the use of closed dielectrophoretic potential cages.The method described teaches how to control the position of eachparticle independently of all the others in a two-dimensional space. Theforce used for trapping in suspension the particles is the negativedielectrophoresis. The individual control on the manipulation operationstakes place through the programming of memory elements and circuitsassociated with each element of an array of electrodes and sensorsintegrated within a same substrate. However, the fixed spatial “noise”bound to the dispersion of the characteristics of the sensors(technically known as “Fixed-Pattern-Noise”) severely limits thereliability of the sensors themselves in the detection of cells. Thepatent reports the use of optical and/or impedance meter sensors. Evenby subtracting a reference image for the compensation of the variationson the gain of the sensors, the result is not completely reliable, sinceother factors, such as for example spatial variations of theillumination (for the optical sensors) or the conductivity of the liquid(for the impedance meter sensors), are not compensated. The U.S. Pat.No. 6,294,063, Becker et al, discloses a method and an apparatus for themanipulation of packages of solid, liquid or gaseous biological materialthrough a distribution of programmable forces. The patent also mentionsthe use of sensors. But also in this case, there is the problem of theFixed-Pattern-Noise.

Another known method for the manipulation of liquid particles (droplets)is the Electro-wetting on Dielectric (EWOD), described in T. B. Jones,Journal of Micromechanics and Microengineering, 15 (2005) 1184-1187. Inthis case, an electric field exerted by electrodes provided on asubstrate allows the propulsion of a droplet surrounded by a gaseousphase in a direction controlled by the sequence of energized electrodes.Devices based on this principle can be made by including a lid (also thesame coated with a dielectric) as teached by the patent application US2004/0058450A1 to Pamula et al., or also simply a wire, called “chain”,which establishes the electric contact with the droplets above thesubstrate. J Berthier et al., NSTI Nanotech 2005, www.nsti.org, vol. 1,2005.

A further force for the manipulation of particles is the viscousfriction force generated by electro-hydrodynamic (EHD) flows, such asthe electro-thermal flows (ETF) or the AC electro-osmosis. In N G.Green, A. Ramos and H. Morgan, J. Phys. D: Appl. Phys. 33 (2000), theEHDs are used for displaying particles. For example, PCT WO 2004/071668A1 describes an apparatus for concentrating particles on someelectrodes, by exploiting the aforesaid electro-hydrodynamic flows.

In “Impedance Spectroscopy Flow Cytometry: On-Chip Label-Free CellDifferentiation”, Cytometry Part A 65A: 124-132 (2005), Cheung K, GawadS, Renuad P, an impedance differential sensor integrated on a microchipis used for differentiating particles in a flow.

In “Near-Field Optical Sensors for Particle Shape Measurements”, IEEESensor Journal, vol. 3, No. 5, October 2003, pp. 646-651, a chip for thedetection of the particle shape based on arrays of integrated sensors(photodiodes) is described. However, the movement of the particles isoperated by a liquid flow, which requires for example a pump or otheranalogous mechanisms, and therefore it is impossible to accuratelycontrol the position of the particles to be analyzed.

In the Italian patent application BO2005A000481, Medoro et al, somemethods for manipulating particles with arrays of electrodes and somemethods and apparatus for their detection are reported, which havehowever limitations similar to the patent PCT/WO 00/69565 alreadymentioned, as for the ability of differentiating the different cells.

Finally, in the International patent application No. PCT/IT02/00524, amethod wherein first biological entities are recognized through secondbiological entities having the capability of binding to the first one(or vice versa) is described, where the first biological entities areimmobilized on a surface defined by a matrix of first electrodes atleast partly selectively activable and addressable, arranged facedtowards at least a second electrode, and are contacted with the secondbiological entities displaced through dielectrophoresis cages; thebonding interaction, if any, between at least a part of the first andthe second biological entities is preferably detected by excitingfluorophores groups bound to the second biological entities withradiations at a first frequency and detecting the fluorescence emissionat a second frequency through optical sensors integrated within theelectrodes. Therefore, there is again the problem of eliminating the“noise” connected with the optical detection (“Fixed Pattern Noise”).

SUMMARY OF THE INVENTION

The aim of the present invention is to provide a method and an apparatusfor carrying out the characterization and/or the counting of particlesof any type, which is free from the drawbacks described and which, inparticular, allows a precision manipulation of the particles to beobtained, being at the same time substantially insensitive to thedrawback of the Fixed-Pattern-Noise.

It is particularly an aim of the present invention to act on the controlof the position of each particle present in the sample, for the purposeof displacing such particles in a deterministic or statistical way, fordetecting their presence and/or characterizing their type withintegrated optical and/or impedance meter sensors.

Here and in the following, by the terms “particles” or “particle”,natural or artificial, micrometric or nanometric entities are intended,such as cells, sub-cellular components, viruses, liposomes, niosomes,microballs (microspheres) and nanoballs, or also smaller entities, suchas macro-molecules, proteins, DNA, RNA, etcetera, as well as drops of anfluid immiscible in a suspension medium, for example oil-in-water orwater-in-oil, or also drops of liquid in a gas (such as water-in-air)or, still, gas bubbles in a liquid (such as air-in-water).

Sometimes, the term cell will be used, but where not otherwise specifiedit should be intended as a non limiting example of the use for thedetection and characterization of particles in the widest sense abovedescribed.

The present invention therefore relates to a method and an apparatus forthe characterization and/or the counting of particles, as abovespecified, according to the claims 1, 3, 5, 9, 11, 20, 22.

In particular, non uniform, time variable force fields and integratedoptical sensors are used. The force fields can be of positive ornegative dielectrophoresis, electrophoresis or electro-hydrodynamicmotions, characterized by a set of stable equilibrium points for theparticles (solid, liquid or gaseous); the same method is adapted to themanipulation of droplets (liquid particles) by exploiting effects knownto the international scientific community by the name of Electrowettingon dielectric.

In this way, the restrictions of the known art are overcome by thepresent invention.

The implementation of the method according to the invention isinsensitive to spatial variations of the illumination and to theFixed-Pattern-Noise. Furthermore, it is possible to accuratelycharacterize and classify the particles manipulated in the devicewithout the need of pumps or liquid flows otherwise generated, whichcause a not well determined positioning of the analyzed particles.

Finally, contrary to the approaches which make use of flows for movingthe particles, by using a controlled force field for moving theparticles relative to the sensor, a greater quantity of information isobtained, consisting for example of the movement speed of the particlein response to the force field. Therefore, an additional informationsource on the characteristics of the particle is obtained. Thisinformation can be advantageously used for differentiating the differenttypes of particles.

Further features and advantages of the invention will be apparent fromthe following description of some non limiting embodiments of the same,which is carried out with reference to the figures of the attacheddrawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the principle of generation of force fields through arraysof electrodes.

FIG. 2 shows the wave forms relating to the method of detection andcharacterization based on a displacement of cages of FIGS. 4 and 9.

FIG. 3 shows microscopically images of (a) a polystyrene ball, (b) a K562 cell, (c) a red cell.

FIG. 4 shows a sequence of steps for the detection/characterization ofparticles with dimensions lower than the electrode.

FIG. 5 shows a diagram of a typical temporal course of the grey valuedetected at the passage of a cell or balls with dimensions in the orderof 15-20 μm, and the indication of some possible discriminantparameters.

FIG. 6 shows the temporal evolution of the grey value detected at thepassage of a K562 cell following to the displacement of its cage.

FIG. 7 shows a diagram of a typical temporal course of the grey valuedetected at the passage of a small cell or microball, and the indicationof some possible discriminant parameters.

FIG. 8 shows the temporal evolution of the grey value detected at thepassage of a polystyrene ball of 10 μm, 6 μm and 3 μm, respectively,following to the displacement of its cage.

FIG. 9 shows a sequence of steps useful for thedetection/characterization of particles with dimensions higher than anelectrode, but lower than two electrodes.

FIG. 10 shows an elemental sequence of steps useful for the simultaneousdetection of all the particles through the movement of all the cages.

FIG. 11 shows three images which can be acquired through theimplementation of the sequence of steps of FIG. 10.

FIG. 12 shows an elemental sequence of steps shown for the simultaneousdetection of all the particles without the need of moving the cages.

FIG. 13 shows the wave forms relating to the detection andcharacterization method based on the activation and the deactivation ofcages of FIG. 12.

FIG. 14 shows another sequence of steps useful for thedetection/characterization of particles in a cage.

FIG. 15 shows the architecture of a device for the high resolutioninvestigation of the features of the particles.

FIG. 16 shows the architecture of a device for the real-timedetermination of the displacement of the particles both in the verticaland the horizontal direction.

FIG. 17 shows some different illumination and optical detection modes.

FIG. 18 shows the top and the sectional view of a special device forpromoting the detection of fluorescent cells, with an emission filterintegrated on the chip surface.

FIG. 19 shows the top and the sectional view of a special device forpromoting the detection of fluorescent cells, with an emission filterintegrated on the internal layers of the chip.

FIG. 20 shows the top and the sectional view of a special device forpromoting the detection of fluorescent cells, with multiple emissionfilters integrated on the chip surface.

FIG. 21 shows a geometrical representation of the counting of differenttypes of particles, particularly of three different types.

DETAILED DESCRIPTION

The aim of the present invention is to provide a method and an apparatusfor carrying out the manipulation and/or the detection of particles.

The method of the invention is based on the use of a non uniform forcefield (F) through which single particles or groups of particles (BEADS)are attracted towards positions of stable equilibrium (CAGE) (FIG. 1).Such field can be for example a negative (NDEP) or a positive (PDEP)dielectrophoresis field (DEP), an electrophoretic field (EF) or anelectro-hydrodynamic (EHD) motions field or still electro-wetting ondielectric (EWOD).

The detection can concern one of the following aspects, or combinationsof the same:

1. the counting of single particles or the quantification;

2. the identification and/or characterization;

3. the localization.

Concerning this, the measurement of the impedance variation and/or themeasurement of the luminous intensity variation transmitted, diffused oremitted in fluorescence is mainly exploited.

Generation of the Forces

There are different methods for the generation of forces for movingparticles, according to the known art (FIG. 1), through arrays ofelectrodes (EL), provided on a substrate. Typically, a cover (LID) isused, which can be in turn an electrode, which delimits a micro-chamberwithin which the particles (BEADS) are typically found in a liquidsuspension. Some diagrams for the different forces are reported inFIG. 1. In case of dielectrophoresis (DEP), the voltages applied are inphase periodic voltages (Vphip) shown with the addition symbol (+) andcounter-phase periodic voltages (Vphin) shown with the subtractionsymbol (−). By counter-phase voltages, voltages out of phase of 180degrees are meant. The field generates a force which acts on theparticles, attracted towards points of equilibrium (CAGE).

In case of negative DEP (NDEP), it is possible to generate closed forcecages, according to the known art, if the cover (LID) is a conductiveelectrode; in this case, the point of equilibrium (CAGE) is provided incorrespondence with each electrode connected with Vphin(−) if theadjacent electrodes are connected with the opposite phase Vphip (+) andif the cover (LID) is connected with the phase Vphin (−). Such point ofequilibrium (CAGE) is normally spaced apart in the liquid relative tothe electrodes, whereby the particles (BEAD) are, in a steady state, inlevitation. In case of positive DEP (PDEP), the point of equilibrium(CAGE) is normally found in correspondence with the surface on which theelectrodes are provided, and the particles (BEADS) are, in a steadystate, contacting with the same. For the PDEP, the presence of furtherelectrodes in the cover is not necessary, because the points ofequilibrium of the PDEP correspond to the maxima of the electric field.An array of electrodes can be used for electrophoresis, in order toattract charged particles towards the electrodes with an oppositepolarity. For the electro-hydrodynamic (EHD) motions, the configurationsof the electrodes generate some flows which drive the particles towardspoints of minimum flow. For the EWOD, a cover (LID) containing adielectric-coated electrode is generally used, and the matrix ofelectrodes is energized by counter-phase signals with respect to thecover in the points in which the particles (typically liquid droplets inair) have to be attracted. The electrodes on which the particle must notbe present, on the contrary, are left floating. For the EWOD, bymanipulating droplets in air, above the array of electrodes, a series ofwires can be used alternatively to the cover.

In the following, for easiness, the use of closed negativedielectrophoresis cages as an execution force for the description of themethods and apparatus of the invention is merely considered by way ofnon limiting example for the purposes of the present invention (for thisreason, a lid acting as an electrode has to be used). To those skilledin the art with ordinary abilities, it is apparent how to generalize themethods and the apparatus described below for the use of differentexecution forces and different types of particles.

Sensors Employed

Always for simplicity, in the following reference will be only made tothe case of the optical sensors, which allow to detect the incidentoptical power on a photodiode integrated with the electrodes. To thoseskilled in the art with ordinary abilities, it is apparent how togeneralize, in the different cases, the methods and apparatus describedbelow also for the alternative or combined use of integrated impedancemeter sensors.

In the following, for simplicity, the term “grey level” will also beused as a synonymous of “exiting signal from the integrated sensor”.This signal, in turn, could be proportional to the “incident opticalpower” (on an optical sensor, such as a photodiode) or to the measuredimpedance (in case of integrated impedance meter sensors).

Methods for the Use of the Optical Sensors

In case of optical sensors, reference will be generally made to the caseof bright-field (or BF)-type illumination. In this case, theillumination strikes on the sensor. Dark-field (or DF) illuminationmethods, or based on fluorescence are also possible and fall within theobject of the present invention, but for easiness they will not belisted for all the methods treated below, but are only discussed herein.

In FIG. 17, some methods for the use of the optical sensors are shown.The figure relates to the section of a device with optical sensors(PIXEL) integrated in a substrate (SUB). The dielectric between thesensors and the different metallizing layers which provide theelectrodes (EL), can simply consist of an oxide (OX) layer or alsoinclude a filter layer (DFL), for example realized with the thin-filmdichroic mirrors technology.

In the diagram of bright-field detection (FIG. 17-BF), the lighting(LIGHT) comes from the cover (LID) and the optical power detected by thesensors is substantially varying as a function of the distortion and theabsorption caused by the particle (BEAD).

In the diagram of dark-field detection (FIG. 17-DF), the lighting(LIGHT) comes through the cover (LID) at such an angle of incidence thatit does not directly reach the sensors (PIXEL), and the optical powerdetected by the sensors is substantially varying as a function of thediffuse radiation (SLIGHT) from the particle (BEAD) hit by the lightingin a direct way (LIGHT), or, in case, following to a reflection on theelectrodes (RLIGHT).

In the diagram of bright-field fluorescence detection (FIG. 17-BFF), theexcitation (EXLIGHT) comes from the cover (LID) and the optical powerdetected by the sensors is substantially varying as a function of theemitted fluorescence (EMLIGHT) by the particle (BEAD). In this case, inorder that the emitted light (EMLIGHT) signal is not overcome by theexcitation power (EXLIGHT), it is advisable to use one or more of thefollowing tricks:

-   -   use a filter layer (DFL) for the wavelength of the radiation        emitted for the excitation;    -   use an excitation frequency for which the optical sensor (PIXEL)        has a low quantum efficiency (i.e. it is relatively insensitive        to that wavelength). This is possible, for example, by using p-n        joints at a relatively high depth, and an excitation radiation        with a wavelength preferably in the UV, in particular better if        lower than 380 nm.

In the diagram of the dark-field fluorescence detection (FIG. 17-DFF),the excitation (EXLIGHT) comes from the cover (LID) at such an angle ofincidence that it does not directly reach the sensors (PIXEL), and theoptical power detected by the sensors is substantially varying as afunction of the fluorescence emitted from the particle (BEAD) hit by thelight in a direct way (EXLIGHT), or, in case, following to a reflectionon the electrodes (RLIGHT). In order that the emitted light (EMLIGHT)signal is not degraded by the power of the light diffused by theparticle (SLIGHT), it is however desirable to use one or more of thetriks above mentioned in case of bright-field fluorescence, so as toimprove the selectivity of the system.

Method for the Detection of the Full Cages Through Activation andDeactivation of the Cages

FIG. 12 shows a possible sequence of steps of the method according tothe present invention, in order to detect which cages are full (at leasta particle is enclosed) and which are empty, without the need ofdisplacing the cages themselves. This method is particularly suitablewhen the pattern applied to the electrodes is so thick not to allow thecages to be displaced. Furthermore, it can be advantageous if in thedevice, circuits for the displacement of the cages are not present.

The method is based on the alternation of execution phases, for theentrapment/release of the particles (BEAD) in the cages (CAGE) andsensing, during which one or more images are acquired by the integratedsensors (PIXEL).

The wave forms are schematized in FIG. 13.

On the deactivation of the cages, the particles start to deposit bysettling, and/or to laterally displace themselves (right part of FIG.12—t_sense) due to the Brownian movements, starting from the position ofstable equilibrium (PEQ) which they assume when caged. Because of thesedisplacements, the grey levels detected in full cages vary in asubstantially higher way with respect to the variations of the greylevels associated with the thermal noise of the reading circuit and/orthe light system, which occur on the sensors (pixel) relating to emptycages.

The classification of empty or full cage takes place as follows:

1. A series of images is acquired (sensing) by alternating operation(activation or deactivation) of the cages (for times in the order ofsome seconds to some fraction of a second) and sensing of one or moreimages (for times in the order of a fraction of a second, e.g. 100 ms,to tens of seconds).

2. For a series of images NIMG, for each sensor PIXEL_IJ associated witha CAGE_IJ, the non-normalized standard deviation of the grey level iscalculated (in this particular case proportional to the incident opticalpower) on the sensor, defined as

${\overset{\sim}{\sigma}}_{{ij} - {NIMG}} = \sqrt{\sum\limits_{k = 1}^{NIMG}\left( {{PIXEL\_ IJ}_{k} - {M\_ IJ}_{NIMG}} \right)^{2}}$with

${M\_ IJ}_{NIMG} = {\frac{1}{NIMG}{\sum\limits_{k = 1}^{NIMG}{PIXEL\_ IJ}_{k}}}$(average value of the grey level of PIXEL_IJ in the series of imagesNIMG).

3. The non-normalized standard deviation average of the grey level onempty reference sensors (PIXEL_REF), and the relative standard deviationof the non-normalized

$M_{{REF} - {NIMG}} = {\frac{1}{NREF}{\sum\limits_{r = 1}^{NREF}{\overset{\sim}{\sigma}}_{r - {NIMG}}}}$standard deviation

$\sigma_{{REF} - {NIMG}} - \sqrt{\frac{1}{NREF}{\sum\limits_{r = 1}^{NREF}\left( {{\overset{\sim}{\sigma}}_{r - {NIMG}} - M_{{REF} - {NIMG}}} \right)^{2}}}$are calculated

4. A classification threshold is defined.THR=M _(REF-NIMG)+α·σ_(REF-NIMG)+Δ. Typical values are α=1,Δ=0.

5. The cages IJ are classified as full, whereby {tilde over(σ)}_(ij-NIMG)>THR and the remaining cages are classified as empty,whereby

It is interesting to {tilde over (σ)}_(ij-NIMG)≦THR. note that suchmethod is independent from the Fixed-Pattern-Noise (fixed spatial noisebound to the dispersion of the characteristics of the photodiodes), asit considers the signal power with respect to the average value persensor, but leaves out the absolute value of such average value.

It is typically sufficient to carry out a number of measurements (thatis to acquire a number of images) NIMG=50-100 in order to obtain anaccuracy higher than 95%.

A dynamic measurement of the number of images NIMG which has to beacquired can be obtained considering the number of images required forconverging the standard deviation (normalized) of the reference pixels

$\sigma_{r - {NIMG}} = \sqrt{\frac{1}{NIMG}{\sum\limits_{k = 1}^{NIMG}\left( {{PIXEL\_ r}_{k} - {M\_ r}_{NIMG}} \right)^{2}}}$to its asymptoticvalue.

This asymptotic value is constant if the noise 1/f bound to the lightvariations is negligible (wherein f is the general frequency in whichthe noise is considered).

Therefore, in more general terms, the method just described with aspecific reference to the case of using optical sensors allows to carryout the detection of the presence of particles (BEADS), if any, existingin points of stable equilibrium (PEQ) of any force field (F) (thereforenot only a dielectrophoretic one) acting on the particles (for examplecells) and generated by an array of electrodes (EL). Such methodincludes the steps of:

i. deactivating the force field (F);

ii. measuring, in at least a time interval following to the deactivationand selected as a function of the dynamic of the settling and/orBrownian movements to which the particles with a deactivated field aresubjected, the grey level generated by first sensors (PIXEL_IJ)associated with points of stable equilibrium (PEQ) and by second sensors(PIXEL_REF) associated with space regions which certainly can not beoccupied by particles because of the current configuration of the forcefield (F);iii. reactivating the force field (F);iv. repeating the steps from i) to iii) a number of times, substantiallycomparable with the convergence rate to its asymptotic value, of thevariance of the grey level values measured on the second sensors,assumed as a reference (PIXEL_REF);v. classifying a point of equilibrium as occupied by particles if, inthe considered temporal series of measurements, the standard deviationof the grey level values detected on the first sensors, associated withsuch point of equilibrium (PEQ), results higher than a pre-fixedthreshold (THR).Method for the Detection of the Full Cages by Movement of Cages andMeasurement of the Difference of the Static Value of the Grey Levels.

FIG. 10 shows the steps of a sequence of operations according to thepresent method for detecting the full cages through a movement of thesame and an analysis of the static values of the grey levels.

1. In a first instant (t0) the cages (CAGE) place the particles (BEADS)on respective sets of initial optical sensors (PIXEL_STA). An image withthe grey levels corresponding with this configuration is acquired.

2. At the following time (t1), once the transient bound to the cagesmovement is exhausted, the particles arrange themselves incorrespondence with final optical sensors (PIXEL_TGT). A new image withthe grey levels corresponding with this configuration is acquired.

3. The difference image of the grey levels in relation to images of thepoints 1 and 2 is determined.

4. The absolute value of the difference image is determined.

5. The grey level resulting for the pairs of pixels (PIXEL_STA,PIXEL_TGT) relating to the starting and final positions of each cage isdetermined.

6. The steps from 1 to 4 are repeated NDIFF times and the grey levelsfor each cage relating to the absolute variation determined at the step5 are accumulated.

7. Analogously to what above described for the classification withactivation/deactivation of the cages, a classification threshold THR isdetermined, considering, this time, average and standard deviation ofthe absolute value of the differences corresponding with certainly emptysensors (pixels) (such as, for example, the pixels between rows ofcages), for all the difference images NDIFF acquired.

In more general terms, the detection of the presence of particles(BEADS) existing, if any, in points of stable equilibrium (PEQ) of aforce field (F) acting on the particles, generated by an array ofelectrodes (EL) takes place by

i) first measuring the grey level generated by first sensors (PIXEL_STA)associated with stable points of equilibrium (PEQ), and by referencesensors (PIXEL_REF) associated with space regions which certainly cannot be occupied by particles in this first current configuration of theforce field (F);ii) next, modifying the force field (F) in order to impart it a secondcurrent configuration wherein the points of stable equilibrium aredisplaced in correspondence with second sensors (PIXEL_TGT), differentfrom the first sensors;iii) next, measuring the grey level generated by the second sensors(PIXEL_TGT) and reference sensors (PIXEL_REF) associated with spaceregions which can not certainly be occupied by particles in the secondcurrent configuration of the force field (F);iv) determining the difference (DIFF_IMG) between the grey level valuesdetected to the preceding points;v) repeating the steps i) to iv);vi) processing differential grey level values in order to classify thepoints of stable equilibrium (PEQ) occupied by particles and those notoccupied.

Such processing includes the steps of vii) determining the absolutevalue of the differences in grey levels, and then viii) classifying asoccupied the points of equilibrium associated with sensors for which avariation substantially higher than the average variation of the greylevel associated with reference sensors which can not be occupied byparticles in the first and second configurations of the field (F) isdetected.

This method, based on the absolute value of image differences, is alsoimmune from the Fixed-Pattern-Noise and, likewise to the method withactivation/deactivation of the cages, is rather insensitive tounevenness in the illumination.

With respect to the first method, it has the disadvantage of requiringcircuits for the displacement of the cages and a distance between thesame which allows such a movement thereof to determine in an univocalway the displacement of the caged cell, if any.

One of the advantages of this method is that a few images (and reducedtimes) are typically required in order to obtain a certainclassification quality (low chances of error) which, with the method ofactivation/deactivation of the cages, requires more images and moretime.

In FIG. 11, the result which can be obtained with a single step(NDIFF=1) is shown. In FIG. 11 (a), the image detectable by the opticalmicroscope is reported, in FIG. 11 (b) the map of the corresponding greyvalues is shown (with a compensation of the Fixed-Pattern-Noise) and onthe contrary, in FIG. 11 (c) the normalized image of the absolute valueof the difference between the image with initial grey values and after adisplacement rightward is shown.

Method for the Detection of the Full Cages Through Movement of Cages andMeasurement of the Dynamic Value of the Grey Levels.

FIG. 2 shows the wave forms in the steps of the methods for the dynamicdetection of the presence of particles in the cages through displacementof the cages themselves.

At the beginning, a displacement of the cages is carried out but withoutwaiting the settling of the particles in their new position ofequilibrium and the signal variations of the optical sensors above whichthe cells (particles) contained in the moving cages are moving, aredetected.

FIG. 4 shows a sequence of steps which involves the movement of the cagein the IJ position and the displacement of the point of equilibrium(PEQ) which determines the passage on the particle (BEAD) on the opticalsensor (PIXEL_IJ).

If the dimensions of the particle (BEAD) are higher than those of theelectrode, the sensor will measure a variation but it does not reach thebase value relating to the pixel without particles above it. In thiscase, a diagram slightly different can be adopted, as in FIG. 9. Thiscase, by measuring the grey value on the pixel (PIXEL_IJ) on the edge ofa cage with higher dimensions than an electrode, reproduces a situationcorresponding to that of having electrodes with double dimensions thanthe actual dimensions of the electrode.

This technique allows, in one sense, to scan the cell by detecting thegrey values associated with the incident power considering the integralwith respect to horizontal sections of the same (if the photodiode is aslarge as the cell and this is vertically moved). By changing theduty-cycle of the execution, a different scanning rate is obtained.

$\frac{t_{act}}{\left( {t_{act} + t_{sense}} \right)}$

These operations allow to detect the presence of particles based on theluminosity peaks and valleys which occur when the cage is full. Byprocessing the signals of the sensors, a person skilled in the art caneasily detect a series of parameters (for example the peak-peakamplitude of the grey value) which allow to discriminate full cages fromempty cages.

Therefore, according to this variant of the method of the invention, thepresence of existing particles (BEAD), if any, in points of stableequilibrium (PEQ) of a force field (F) acting on the particles,generated by an array of electrodes (EL) is detected by carrying out thefollowing steps:

i. modify the force field (F) so as to move the points of stableequilibrium (PEQ) by passing the particles (BEADS) in correspondencewith respective sensors (PIXEL_IJ);

ii. measuring the grey level generated by sensors (PIXEL IJ) associatedwith the passage of the different points of stable equilibrium (PEQ)with a substantially higher frequency than the settling time of theparticles in the new position of equilibrium;

iii. classify the presence or the absence of particles through themeasurement of characteristic parameters of the temporal evolution ofthe measurement of the grey level generated by the sensors, for examplebased on the incident optical power, in case of optical sensors.

The measurement of the grey level generated by the sensors, namely theoptical power striking on the same, can obviously take place alsoalternating the steps of activation of the field acting on the particle;and deactivation of the aforesaid field, in order to measure,afterwards, the incident optical power, that is the grey level thusobtained; this is advantageous for avoiding any interference between theexecution voltages with the reading of the sensors, even if in principleit is not necessary if there is a substantial independence of thesensors from the activation of the execution voltages.

The parameters of the temporal evolution of the measurement of theoptical power preferably include the peak to peak amplitude of the greylevel, and preferably the classification takes place through thecomparison with a threshold value determined starting from peak to peakmeasurements of the optical power, namely the grey level, on referencesensors which can not be occupied by particles with the configuration ofthe considered force field (F), increased by a factor proportional tothe standard deviation of the peak to peak values of the referencesensors.

In this way, also the compensation of the noise 1/f due to theenvironment illumination existing on the reference sensors is obtained.

Method for the Counting of Cells Based on the Number of Full Cages, withError Compensation

By the methods above described, the number of full cages is thendetermined. The aim of the present invention is also to find out amethod for the counting of the cells in the cages. From this, the volumeof the considered sample being known, also the concentration of thecells is obtained.

As a first approximation, especially if the average number of cells percage is substantially lower than about 0.1, the number of full cages isapproximately similar to the number of cells (in this case there is anunderevaluation of the number of cells of about 5%). For a higheraverage number of cells, according to the present invention, thedistribution statistics of the cells per cages can be compensated, inorder to obtain a counting which approximates with a greater precisionto the actual number of cells on the chip.

We assume that the following suppositions occur:

1. The cells are uniformly distributed per volume unit; this hypothesisis overall verified in the cases in which the sample is injected in theempty micro-chamber.

If, on the contrary, the sample is injected in the micro-chamberpreviously filled of buffer, the sample could be only locally uniform,because of the variations of cells density bound to the flow profile ofthe sample.

2. The bonds connected with the maximum number of cells per cage can beignored.

This hypothesis is verified when the total volume of the cells (averagevolume of a cell per average number of cells) is reasonably lower thanthe volume of a cage (intended as the volume of the basin of attractionof each cage).

Under these hypothesis, the statistical distribution of the cells isrepresented by the binomial distribution. We define:

NCAGES=number of cages in the chip (or considered portion)

n=NCELLS=number of cells in the chip (or considered portion)

wherein by “chip” the set formed by the array of electrodes EL isintended, which is precisely normally integrated in a single multilayerchip together with the sensors PIXEL.

Then, the probability that a cell belongs to the basin of attraction ofany cage is:p=1/NCAGES

The probability of having k cells per cage results from the formula:

${P\lbrack k\rbrack} = {\begin{pmatrix}n \\k\end{pmatrix} \cdot p^{k} \cdot \left( {1 - p} \right)^{n - k}}$${{wherein}\mspace{14mu}{is}\mspace{14mu}\begin{pmatrix}n \\k\end{pmatrix}} = \frac{n!}{{k!}{\left( {n - k} \right)!}}$the number of combinations of n objects taken k at a time (binomialdistribution).

The average value of cells per cage (Average Cells Per Cage—ACPC) is

${ACPC} = {{< {P\lbrack k\rbrack}>={p \cdot n}} = \frac{NCELLS}{NCAGES}}$

As on the chip there are typically many cages, the probability of having1, 2, . . . k cells per cage, multiplied by the number of cages,approximates well the actual value of cages with 1, 2, . . . k cells.Considering this, and the total number of empty and full cages beingdetected, the average value of cells per cage (ACPC) can be estimated,by calculating the value which provides an expected number of full andempty cages corresponding with the detected value.

The expected value of empty cages is calculated as

$\quad\begin{matrix}{{EC}_{estimate} = {{NCAGES} \cdot {P\lbrack 0\rbrack}}} \\{= {{NCAGES} \cdot \left( {1 - p} \right)^{n}}} \\{= {{NCAGES} \cdot \left( {1 - \frac{1}{NCAGES}} \right)^{NCELLS}}}\end{matrix}$and by replacing the value of the empty cages actually detected, thecounting of cells is then obtained, which can be calculated with simplepassages such as

${NCELLS}_{measure} = \frac{{\log({NCAGES})} - {\log\left( {EC}_{measure} \right)}}{{\log({NCAGES})} - {\log\left( {{NCAGES} - 1} \right)}}$

A better estimation of the number of cells is thus obtained, and theprecision of the counting results good also for ACPC values higher thanone, provided that the number of empty and full cages is statisticallysignificant; in this way, in fact, the error bound to theunderevaluation of the number of cells otherwise associated with thecounting of the full cages alone is mitigated. Obviously, the detectednumber of empty cages (EC_(measure)) must be greater than zero andresults lower or equal to the number of cages NCAGES. The followingtable numerically shows the case for a particular example with 6400cages, and different concentrations of cells. As it can be noted, thecounting error is drastically reduced, especially for high averageconcentrations of cells per cage. Actually, the reported value is simplythe one based on the density of probabilities, but given the greatnumber of cages, the probability estimation approximates well a possiblemeasured value.

TABLE 1 NCAGES 6400 6400 6400 6400 NCELLS 640 3200 6400 12800 ACPC 0.1000.500 1.000 2.000 k P[k] cages cells P[k] cages cells P[k] Cages cellsP[k] cages cells 0 90.48% 5791 0 60.65% 3882 0 36.79% 2354 0 13.53% 8660 1 9.05% 579 579 30.33% 1941 1941 36.79% 2355 2355 27.07% 1732 1732 20.45% 29 58 7.58% 485 970 18.40% 1177 2354 27.07% 1732 3464 3 0.02% 1 31.26% 81 243 6.13% 392 1176 18.05% 1155 3465 4 0.00% 0 0 0.16% 10 401.53% 98 392 9.02% 577 2308 5 0.00% 0 0 0.02% 1 5 0.31% 20 100 3.61% 2311155 6 0.00% 0 0 0.00% 0 0 0.05% 3 18 1.20% 77 462 7 0.00% 0 0 0.00% 0 00.01% 0 0 0.34% 22 154 8 0.00% 0 0 0.00% 0 0 0.00% 0 0 0.09% 5 40 90.00% 0 0 0.00% 0 0 0.00% 0 0 0.02% 1 9 10 0.00% 0 0 0.00% 0 0 0.00% 0 00.00% 0 0 11 0.00% 0 0 0.00% 0 0 0.00% 0 0 0.00% 0 0 12 0.00% 0 0 0.00%0 0 0.00% 0 0 0.00% 0 0 13 0.00% 0 0 0.00% 0 0 0.00% 0 0 0.00% 0 0 140.00% 0 0 0.00% 0 0 0.00% 0 0 0.00% 0 0 15 0.00% 0 0 0.00% 0 0 0.00% 0 00.00% 0 0 total 609 640 2518 3199 4045 6395 5532 12789 counting error−4.84% 0.00% −21.31% −0.03% −36.80% −0.08% −56.78% −0.09%

In other words, according to this method, from the counting of full andempty cages, carried out according to any one of the method described sofar, the counting of the total number of particles (for example cells)actually existing in a sample injected in a chamber defined by the arrayof electrodes EL and the cover (LID), if any, can be derived; thecounting step of the single particles, of which the general presence hasbeen previously detected, takes place according to this aspect of theinvention in an estimated way on statistical bases, as follows:

a) the number (EC_(measure)) of points of stable equilibrium (PEQ)existing in the field (F) generated by the array of electrodes(EL)—points of stable equilibrium, hereinbelow also shown as—cages—whichdo not contain any particle (BEAD) is measured by the sensors.

b) The counting (NCELLS) of particles (BEAD) is determined as the ratioof the difference between the logarithm of the number (NCAGES) of pointsof stable equilibrium (PEQ) to the logarithm of the measured number(EC_(measure)) of the points of stable equilibrium (PEQ) non containingany particle, and the difference between the logarithm of the number(NCAGES) of points of stable equilibrium (PEQ) and the logarithm of thenumber (NCAGES−1) of the points of stable equilibrium (PEQ) less one.

Method of Characterization of the Cells in the Cages Through Movement ofthe Cages and Measurement of the Dynamic Value of the Grey Levels.

As it is shown in FIG. 3, since a ball (ex. FIG. 3 (a)), a cell of atype (ex. a K562, as in FIG. 3 (b)), or a cell of another type (ex. ared cell as in FIG. 3 (c)), have different dimensions, shape, absorbanceprofile and index of refraction, by passing the cage on a sensor it ispossible to detect not only if it is full but also, according to afurther aspect of the present invention, to characterize the type of theexisting particle, if any.

A cell (and certain types of micro-balls) show a light distortion (akind of a lens effect) which causes a variation of the grey level withnegative peaks (dark edges on the cell) and positive peaks (brightercentre where the cell concentrates the light) of the grey level.

Parameters bound to the dynamic evolution of the grey level, such as, bya significant but not limiting example reported in FIG. 5, themeasurement of the amplitude of the negative (Gnp) and positive (Gpp)peaks of the grey level, as well as for example the delay (td) betweencage movement and detection of the first grey variation, or also thetemporal length of the variations can be used for obtaining additionalinformation and classifying the kind of cell.

Formulas:

The displacement speed is, as a first approximation, proportional to thedielectrophoretic mobility, and results v∝k·R². The length of thevariation of the grey levels, as a first approximation, thereforeresults, namely inversely proportional to the radius of the particle (itdecreases when R increases). The time delay td between

${t_{w} = {\frac{2\; R}{{kR}^{2}} \propto \frac{1}{R}}},$execution and start of the variation of the grey level results as afirst approximation (in the case of FIG. 4, with a cell on a singleelectrode) equal to(it also decreases when R increases).

FIG. 6 shows the actual course measured for a K562 cell.

$t_{d} = \frac{W_{EL} - R}{{kR}^{2}}$

FIG. 7 shows the typical course expected for non transparent balls, orsmall cells for which the effect of the absorbance compared to the abovementioned “lens” effect is predominant.

FIG. 8 shows the temporal evolution of the grey value detected at thepassage of polystyrene balls with 10 μm, 6 μm and 3 μm following to thedisplacement of its cage.

By extracting opportune discriminating parameters, it is possible, in anapparent way for those skilled in the art, to define a classificationcriterion, similarly to what above mentioned for the classification offull and empty cages.

In particular, the identification method can include the extraction ofthe discriminating characteristics of the particles, and the use ofalgorithms based on neural networks, first close ks, thresholdsalgorithms and/or Principal Component Analysis, or a combination of thesame.

FIG. 14 shows an alternative technique based on the same principle,through which it is possible to scan the cell with a greater resolutionin its horizontal section, by laterally moving it above the sensor. Infact, the “slice” of the considered cell has lower dimensions. From thederivative of the grey value as the cell goes by, the punctual value ofthe grey value along a horizontal section of the cell can be calculated,with a horizontal resolution equal to the number of points during theacquisition, and vertical resolution equal to the gap (distance) betweenthe electrodes.

Based on what above described, it is therefore apparent that theinvention also allows to characterize particles (BEADS) (that is, forexample, to establish their physical nature) present in points of stableequilibrium (PEQ) of a force field (F) acting on the particles andgenerated by an array of electrodes (EL), through the carrying out ofthe following steps:

a. modifying the force field (F) so as to displace said points of stableequilibrium (PEQ) by passing said particles (BEADS) in correspondencewith respective sensors (PIXEL_IJ);

b. measuring the grey level detectable by the sensors (PIXEL_IJ)associated with the transit of different points of stable equilibrium(PEQ) containing particles, with a frequency substantially higher thanthe settling time of the particles in the new equilibrium position;c. processing characteristic parameters of the temporal evolution of thegrey level measurement for estimating the nature of the particles.

The whole always without being affected from the background “noise”detectable by the sensors, namely the Fixed Pattern Noise.

In the described example, where the sensors used are optical sensors andthe measurement of the grey level is a measurement of the optical powerstriking on the sensors, such measurement preferably takes place byalternating the steps of

i. activating the field acting on the particle;

ii. deactivating the field and measuring the incident optical power; orby using the same preferred system above described for the dynamicdetection of the presence of particles in the cages. The parameters ofthe temporal evolution of the measurement of the optical power include,based on what above described, at least one of the parameters selectedfrom the group consisting of:i. the delay (td) between the variation of the force field (F) and thefirst variation of the optical powerii. the length (tw) of the transitory variation of the optical power;iii. the amplitude of the positive peak (Gpp) of the optical powervalue;iv. the amplitude of the negative peak (Gnp) of the optical power value.Method for the Counting of Different Species of Cells in anHeterogeneous Sample

By combining the methodologies for the counting of cells for a singlepopulation with the characterization of the particles in the singlecages it is possible, according to the present invention, to determinethe composition of a sample with heterogeneous types of particles.

Generally, it is not easy to differentiate the composition of particlesin cages which contain more than one cell. However, by processing thedynamic of the grey level, recorded as above described for thecharacterization of the content of a cage, it is relatively simple todetermine if the cage contains a single particle or a multiplicity ofparticles (MC).

Since it can be assumed that the distribution of different types ofparticles are independent (orthogonal) therebetween, by combining thenumber of particles of each type obtained in the cages with a singleparticle, and the information relating to the number of empty cages (EC)and the total number of cages, it is therefore possible to determine theestimation of the number of particles of the different populations, by anumerical route. The problem is the minimization of a function withvarying NPT (where NPT=number of types of particles). In FIG. 21 arepresentation of the problem in case of NPT=3 is reported.

For each type of particle t=1, . . . NPT, once the presence of a numberof particles (NCELLSt) has been supposed, the volume of the hypercube ofthe space to NPT dimensions which represents the number of particles ofthe t type existing in a cage alone (by ignoring the other kinds ofparticles), remains in fact fixed (Pt_1). With reference to the FIG. 21(a), for the particles of a first type, we will obtain P1_1 particles ina single cage, P1_M particles in multiple cages and P1_0 cages withoutparticles of the type P1 (the measures of the shown volumes). Thedetected number of t-type particles in a single cage must take intoaccount, however, that one or more particles of other types can bepresent in the same cages. Therefore, as shown in FIG. 21( d), thedetected value of single particles (Pt_1_MEAS) of each type tcorresponds to the volume (Pt_1) of the hypercube of particles of type tminus the volume of the intersection hypercubes between Pt_1 and Pq_h,with q< >t and h>0 of hypercubes which contain at least a particle of adifferent type. The volume of the hypercube of empty cages (EC_MEAS) isfurther known.

The tuple of values NCELLSt, t=1, . . . , NTP is calculated by numericalway, whereby the (statistically) expected value better corresponds tothe actually measured number (Pt_1) of cages with a single particle ofeach type t and of empty cages (EC_MEAS).

Based on what just described, it is therefore apparent that theinvention also allows to count the number of particles (BEADS) of amultiplicity of types (NTP), existing in points of stable equilibrium(PEQ) of a force field (F) acting on the particles and generated by anarray of electrodes (EL), through the execution of the following steps:

a. modifying the force field (F) so as to displace said points of stableequilibrium (PEQ) by passing said particles (BEADS) in correspondencewith respective sensors (PIXEL_IJ);

b. measuring the grey level detectable by the sensors (PIXEL_IJ)associated with the passage of the different points of stableequilibrium (PEQ) containing particles, with a frequency substantiallyhigher than the settling time of the particles in the new equilibriumposition;c. processing characteristic parameters of the temporal evolution of thegrey level measure associated with each point of stable equilibrium(PEQ) for detecting if the associated cage is empty, if it contains aparticle of a type or if it contains more particles;d. determining in a numerical way the combination of countings ofparticles of each type (NCELLSt) which better approximates to themeasured value of cages with a single particle of each type (Pt_1_MEAS)and empty cages (EC_MEAS).Apparatus for the High Resolution Characterization of the Cells in theCages Through Movement of the Cages and Measurement of the Dynamic Valueof the Grey Level.

In order to increase the resolution of the dynamic detection methodsdescribed so far, according to the present invention it is possible tocombine (FIG. 15) a series of electrodes (EL) associated withphotodiodes (PIXEL_V1, . . . PIXEL_VN) characterized by a greaterspatial resolution, to sequentially analyze different sections of theparticle, by sequentially moving it on different electrodes.

The resolution limit is thus determined by the minimal geometry of thephotolithographic resolution and it does not have the need of placingdifferent transistors for the addressing of the electrodes (pixels),which would increase the required area. For example, some openings(SLITS) can be carried out on a metallizing arranged upon thephotodiode, so as to render it sensitive only in correspondence with thesame opening.

In this way, a further step can be implemented in all the methodsdescribed so far, in which said points of stable equilibrium (PEQ) aredisplaced such that said particles (BEADS) are passed in correspondencewith a plurality of respective said sensors (PIXEL_IJ); and in which thesensors (PIXEL_IJ) are optical sensors and are shielded (through theaforesaid metallizing layer equipped with openings SLITS of a pre-fixedand relatively small amplitude), so as to detect with the sensors theincident optical power on a space portion substantially lower than thedimensions of the particles and the sensors themselves.

Method for the Manipulation of Cells with an Automatic Control of theDisplacement Speed of the Cages

A method of manipulation with an automatic control of the displacementspeed of the cages can then be based on the use of the passage detectionof the cells, as above described. For the cages with cells which aremoving on the chip (together with the array of electrodes) the evolutionof the grey levels can be monitored and the end of the settlingtransient in the new point of equilibrium can be detected forimmediately starting the following step along the trajectory. Besidesthe optimization of the times, by dynamically adapting them to the speedof the different particles in the different moments, such approach alsoallows to verify that each cell does not remain blocked.

If to each electrode a single optical sensor is associated, for examplein the vertical gap (distance) between two electrodes, as in FIG. 4,this method cab be carried out using cages with a width of 1×2electrodes (1 in a horizontal position×2 in a vertical position). In thehorizontal displacement, in fact, the particle locates itself at fullspeed on the sensor between the two electrodes, and its movement can befollowed by monitoring the sensor in correspondence with the initialand/or final point of equilibrium. This introduces a bond on the cageshape, which however can be overcome by using an apparatus as describedbelow.

Apparatus for the Manipulation of Cells with Automatic Control of theDisplacement Speed of the Cages, without Bonds on the Dimensions of theSame.

In FIG. 16, the diagram of an apparatus with sensors (pixels) for themovement determination both in horizontal (PIXEL_H) and in vertical(PIXEL_V) direction is shown. Such apparatus is advantageous forimplementing a manipulation of particles with a close chain control,carried out in an integrated manner with the one of the displacementspeed of the cages. With this apparatus, the horizontal displacementalso with cages 1×1 can be carried out, since the cell/particle passesin any case on the sensor (PIXEL_H) placed between two electrodes alongthe horizontal axis.

With the additional method and apparatus now described, a stepapplicable on all the methods of detection and characterization ofparticles above described is implemented, consisting of the control ofthe variation course in the grey level detectable by the sensors(PIXEL_IJ) associated with the passage of the points of stableequilibrium (PEQ) containing particles, so as to determine the end ofthe settling transient of the particles in the new position ofequilibrium and go back from this measure to the displacement speed ofthe particles (BEADS) towards new points of stable equilibrium (PEQ). Atthe end of such a settling transient, a variation in the force field (F)produced by the electrodes themselves can be therefore automaticallygenerated (for example through a proper software implemented in acontrol unit CTRL, moreover of a known type, of the array of electrodesEL (FIG. 15), for the purpose of causing a new displacement of thepoints of stable equilibrium (PEQ) along the pre-fixed desiredtrajectory (shown in FIG. 15 by the arrow).

Apparatus with Integrated Dichroic Filters for the Detection and/or theCharacterization of the Cells in the Cages by Means of Fluorescence

For the fluorescence detection and/or the characterization of cells,some preferred implementations according to the present invention arereported. These implementations integrate a filter element which allowsthe passage of the fluorescence emission but mitigates the excitation.These filter elements can be integrated with process steps downstream ofthe chip execution, such as thin film depositions of materials withopportune indexes of refraction for carrying out dichroic filters. Thefilter effect allows to mitigate the part of optical power in theexcitation band detected by the sensor (PIXEL), thus reducing therequirements bound to the dynamic range of the sensor itself.

In FIG. 18, the diagram of an apparatus particularly suitable for thedetection and/or characterization of fluorescence cells is shown. Adichroic filter (DFL) which allows the passage of the frequenciescorresponding to the emission is deposited, with techniques known tothose skilled in the art, on the chip. After the deposition, optionally,wishing to improve the electric contact between an electrode (EL) and aliquid, some windows on the dichroic layer are opened, in correspondencewith the electrodes themselves. In this way, it is possible, forexample, to use even high conductive solutions, in case ofelectrophoresis, without voltage drops connected with the presence ofthe dielectric layer composed of the dichroic filter. This executionscheme is compatible with the use of a standard CMOS wafer manufactureand only involves a post-processing of the wafers.

Alternatively, the dichroic filter can be carried out below theelectrodes, as it is shown in FIG. 19. This can be useful if the CMOSprocess can be modified. Also without the modification of the CMOSprocess, this diagram can be however carried out by performing apost-processing, even if more complex, represented by the application ofthe filter, the opening of contacts at the CMOS top metal and theadditional metallizing for carrying out the electrodes (EL). It can beadvantageous to carry out, regardless by the fact of being above orbelow the electrodes (EL), a spatially organized multiplicity ofdichroic filters with a different passband (DFL1, DFLN), as it is shownin FIG. 20. This can be useful for dividing and separately detecting thefluorescence of cells/particles at different emission frequencies.

Clearly, by checking the cells position it is then possible to analyzethem in sequence, on different sensors, for the presence for example ofdifferent fluorophores. According to this aspect of the invention justdescribed, the invention relates to an apparatus for the detectionand/or characterization of particles as above described, but furtherincluding shielding means of the sensors and openings, in a number of atleast one for each sensor, obtained through the shielding means andhaving pre-fixed dimensions, smaller than those of the particles to bedetected/characterized.

The apparatus according to the invention can further include at least adichroic filer with a pre-fixed passband arranged such that it shieldsat least partly the sensors (PIXEL), if these are optical sensors, andpreferably include a plurality of dichroic filters, each having apassband different from the others, arranged superimposed therebetweenand so as to shield however at least partly the sensors (PIXEL). If atleast a dichroic filter is arranged to cover the electrodes (EL), it isequipped with interruption openings placed in correspondence with atleast part of each electrode.

Filtering of the Excitation

The apparatus can take advantage, or not, of the fact that (externally)the excitation source is filtered and optimized for each fluorophore tobe analyzed. This can be easily carried out with filters non integratedon the device. Furthermore, or alternatively, the lid of the chip canintegrate the part of the excitation filtering corresponding withunderlying dichroic filters, which vary, or not, from pixel to pixel ofthe chip.

If the excitation filtering is integrated on the lid, it is necessary tocarry out wider zones with a homogeneous dichroic filter, so as to avoidcross-talk between the excitations of the different pixels. In thiscase, the cell under test must cover a greater distance for beinganalyzed for different fluorophores. According to this additional aspectof the invention, the apparatus described so far can therefore includeat least a dichroic filter with a pre-fixed passband arranged incorrespondence with the cover (LID) defining, together with the array ofelectrodes (EL), a chamber or micro-chamber suitable for receiving andcontaining a fluid sample containing the particles to bedetected/characterized.

Compensation Method of the Illumination Noise

In all the methods above mentioned, when optical sensors are used, thesignal for the detection or the characterization of the particlesdepends on the illumination power. Therefore, variations of this typecan cause some variations of the level of the optical power detectedwhich are not bound to the signal (presence and/or position of theparticle). Although normally this is not a problem, the performances ofthe methods (greater precision, greater speed) can be however improved,compensating such variations by using a value normalized to the averagedetection of the reference pixels (pixels certainly corresponding withempty cages, for which the optical incident power is only affected bythe illumination power).

This is mainly true for the low-frequency illumination noise, whichbecause of the spectral density of the noise power (proportional to 1/f,makes the impact thereof more influent.

Method of Noise Compensation for Impedance Sensors.

In all the methods above mentioned, when impedance meter sensors areused, the signal for the detection and the characterization of theparticles depends on the conductivity and permittivity of the suspensionmedium of the particles, which in turn depends for example on thetemperature, the salts concentration or other molecules, etcetera.Therefore, variations of these can cause some variations of the level ofthe detected impedance which are not bound to the signal (presenceand/or position of the particle). Although normally this is not aproblem, the performances of the methods (greater precision, greaterspeed) can be however improved, compensating such variations by using avalue normalized to the average detection of the reference pixels(pixels certainly corresponding with empty cages, for which theimpedance is only affected by the conductivity and the permittivity ofthe suspension medium).

In all the methods of detection and characterization described so far, afurther step consisting of a compensation of the grey levels detected bysaid sensors (PIXEL_IJ) can therefore be introduced, using a valuenormalized to the average detection of reference sensors (PIXEL_REF)associated with space regions which can not certainly be occupied byparticles in the current configuration of the considered force field(F).

Note on the Applications

The methods and the apparatus described so far are of a general use andfind multiple applications. By way of example but not limitation of theinvention, we mention some of the most important applications:

-   -   Analysis of blood samples; by introducing a sample on the chip        (which uses for example closed dielectrophoresis cages) the red        cells can be counted by counting as a first approximation all        the occupied cages (in fact the other cells possibly existing        have a so lower concentration that it results negligible for the        precision typically required).    -   Emochroma: characterization of the number and type of cells in        the blood; in this case, the method above described for carrying        out an accurate characterization of each single existing cell is        employed, by classifying it as a red cell, platelet lymphocyte,        etcetera.    -   Counting of the bacteria existing in a sample; by injecting on        the chip (with DEP closed cages) a known volume of sample, all        the bacteria existing are detected, and if necessary also the        type, if they are distinguishable through their characteristic        signature detected with the optical sensors.    -   Separation/counting of the cells labelled with fluorescence from        a mixed population. This type of problem is widely diffuse both        in the research and the diagnostics.

For example, bacteria in dairy samples, such as milk, yoghurt, ice-creampreparations, etcetera can be detected.

As another example, bacteria for a fermentation of grape must (forexample for enological applications) can be detected.

As a further example, bacteria existing in a volume of drinking watercould be detected.

The invention claimed is:
 1. Method for the detection of the presence ofparticles (BEADS) possibly present in points of stable equilibrium (PEQ)of a force field (F) acting on said particles, generated by an array ofelectrodes (EL), comprising the steps of: i. deactivating the forcefield (F); ii. measuring, in at least a time interval following thedeactivation, a grey level generated by first sensors (PIXEL_IJ)associated with said stable equilibrium points (PEQ) and by secondsensors (PIXEL_REF) associated with space regions not occupied byparticles because of the current configuration of said force field (F),wherein the time interval is a function of a dynamic of settling and/orBrownian movements to which the particles are subjected with thedeactivating of the force field; iii. reactivating the force field (F);iv. repeating the steps from i) to iii) a number of times, wherein thenumber of times is substantially comparable with convergence rate, ofthe variance of the grey level measured on said second sensors to itsasymptotic value; v. classifying a point of equilibrium as occupied byparticles if, in the considered temporal series of measurements, thestandard deviation of the grey level measured on the first sensors ishigher than a pre-fixed threshold (THR).
 2. Method according to claim 1,characterized in that said threshold (THR) corresponds to the averagevalue of the grey level detected on said second sensors increased by afactor proportional to the standard deviation of the grey level detectedon said second sensors (PIXEL_REF).
 3. Method for the detection of thepresence of particles (BEADS) possibly existing in points of stableequilibrium (PEQ) of a force field (F) acting on said particles,generated by an array of electrodes (EL), comprising the steps of: i.measuring a grey level generated by first sensors (PIXEL_STA) associatedwith said points of stable equilibrium (PEQ), and by reference sensors(PIXEL_REF) associated with space regions not occupied by particles in afirst current configuration of said force field (F); ii) modifying saidforce field (F) in order to impart it a second current configurationwherein said stable equilibrium points are displaced in correspondencewith second sensors (PIXEL_TGT), different from the first sensors; iii)measuring a grey level generated by the second sensors (PIXEL_TGT) andby reference sensors (PIXEL_REF) associated with space regions notoccupied by particles in said second current configuration of said forcefield (F); iv) determining the difference (DIFF_IMG) between the greylevel detected in steps i) and iii); v) repeating the steps i) to iv);vi) processing differential grey level values in order to classify thepoints of stable equilibrium (PEQ) occupied by particles and those notoccupied.
 4. Method according to claim 3, characterized in that suchprocessing includes the steps of determining the absolute value of thedifferences in grey levels, and classifying as occupied the points ofequilibrium associated to sensors for which a variation substantiallyhigher than the average variation of the grey level associated with saidreference sensors which can not be occupied by particles in said firstand second configurations of the force field (F) is detected.
 5. Methodfor the detection of the presence of particles (BEADS) possibly existingin points of stable equilibrium (PEQ) of a force field (F) acting onsaid particles, generated by an array of electrodes (EL), comprising thesteps of: i. modifying said force field (F) so as to displace saidpoints of stable equilibrium (PEQ) to get said particles (BEADS) pass incorrespondence with respective sensors (PIXEL_IJ); ii. measuring a greylevel generated by sensors (PIXEL_IJ) associated with the passage ofsaid different points of stable equilibrium (PEQ), with a frequencysubstantially higher than the settling time of said particles in the newequilibrium position; iii. classifying the presence or the absence ofparticles through the measurement of at least a characteristic parameterof the temporal evolution of the grey level measured by said sensors(PIXEL_IJ).
 6. Method of claim 5, characterized in that saidcharacteristic parameter of the temporal evolution of the measurement ofthe grey levels on said sensors (PIXEL_IJ) comprises the peak to peakamplitude.
 7. Method of claim 5, characterized in that theclassification takes place through the comparison with a threshold valuedetermined starting from peak to peak measurements of the grey levels onreference sensors which cannot be occupied by particles with theconfiguration of the considered force field (F), increased by a factorproportional to the standard deviation of said peak to peak values ofsaid reference sensors.
 8. Method of claim 5, further including acounting step of the total number of particles of which the presence hasbeen detected, characterized in that said counting step takes place in away estimated on statistical bases, in the following way: i. the number(EC_(measure)) of points of stable equilibrium (PEQ) existing in thefield (F) generated by the array of electrodes (EL), which do notcontain any particle (BEAD), is measured by the sensors; ii. thecounting (NCELLS) of particles (BEADS) is determined as the ratio of thedifference of the logarithm of the number (NCAGES) of points of stableequilibrium (PEQ) to the logarithm of the measured number (EC_(measure))of the points of stable equilibrium (PEQ) non containing any particle,and the difference of the logarithm of the number (NCAGES) of points ofstable equilibrium (PEQ) to the logarithm of the number (NCAGES-1) ofthe points of stable equilibrium (PEQ) less one.
 9. Method for thecharacterization of particles (BEADS) existing in points of stableequilibrium (PEQ) of a force field (F) acting on said particles,generated by an array of electrodes (EL), comprising the steps of: i.modifying said force field (F) so as to displace said points of stableequilibrium (PEQ) by passing said particles (BEADS) in correspondencewith respective sensors (PIXEL_IJ); ii. measuring a grey level generatedby sensors (PIXEL IJ) associated with the passage of the differentpoints of stable equilibrium (PEQ) containing particles, with afrequency substantially higher than the settling time of said particlesin the new position of equilibrium; iii. processing characteristicparameters of the temporal evolution of the grey level measurement inorder to estimate the nature of said particles.
 10. Method of claim 9,characterized in that said parameters of the temporal evolution of thegrey level include at least one of the parameters selected from thegroup consisting of: i. the delay (td) between the variation of theforce field (F) and the first variation of the grey level; ii. thelength (tw) of the transitory variation of the grey level; iii. thewidth of the positive peak (Gpp) of the grey level; iv. the width of thenegative peak (Gnp) of the grey level.
 11. Method for counting thenumber of particles (BEADS) of a multiplicity of types (NTP), existingin points of stable equilibrium (PEQ) of a force field (F) acting onsaid particles and generated by an array of electrodes (EL), comprisingthe following steps: i. modifying the force field (F) so as to displacesaid points of stable equilibrium (PEQ) by passing said particles(BEADS) in correspondence with respective sensors (PIXEL_IJ); ii.measuring a grey level detectable by the sensors (PIXEL_IJ) associatedwith the passage of the different points of stable equilibrium (PEQ)containing particles, with a frequency substantially higher than thesettling time of the particles in the new position of equilibrium; iii.processing characteristic parameters of the temporal evolution of thegrey level measurement associated with each point of stable equilibrium(PEQ) for detecting if the associated cage is empty, if it contains aparticle of a type or if it contains more particles; iv. determining ina numerical way the combination of countings of particles of each type(NCELLSt) which better approximates to the measured value of cages witha single particle of each type (Pt_1_MEAS) and empty cages (EC_MEAS).12. Method of claim 11, characterized in that said points of stableequilibrium (PEQ) are displaced such that said particles (BEADS) arepassed in correspondence with a plurality of respective sensors(PIXEL_IJ); and in that said sensors (PIXEL_IJ) are optical sensors andare shielded so as to detect the incident optical power on a portion ofspace substantially lower than the dimensions of the particles. 13.Method of claim 12, characterized in that the course of the variation inthe grey level detectable by the sensors (PIXEL_IJ) associated with thetransit of said points of stable equilibrium (PEQ) containing particlesis controlled, so as to determine the end of the settling transient ofsaid particles in the new equilibrium position and go back from thismeasurement to the displacement speed of said points of stableequilibrium (PEQ).
 14. Method of claim 13, characterized in that at theend of said settling transient, a variation in said force field (F) isproduced for causing a new displacement of said points of stableequilibrium (PEQ).
 15. Method of claim 1, characterized in that saidsensors are optical sensors and said grey level corresponds with theoptical power striking on the sensors.
 16. Method of claim 1,characterized in that said sensors are impedance meter sensors. 17.Method of claim 1, characterized in that a compensation of the values ofthe grey levels detected by said sensors (PIXEL_IJ) is carried out usinga value normalized to the average detection of reference sensors(PIXEL_REF) associated with space regions which cannot certainly beoccupied from particles in said current configuration of said forcefield (F).
 18. Method of claim 1, characterized in that said measurementof the grey level takes place by temporarily deactivating said forcefield (F).
 19. Method of claim 1, characterized in that said force field(F) is generally selected from: positive dielectrophoresis, negativedielectrophoresis, Electrowetting on dielectric, electrohydrodynamicflows, electrophoresis.
 20. A method for the detection and/orcharacterization of particles (BEADS) comprising generating points ofstable equilibrium (PEQ) of a force field (F) acting on said particlesby an array of electrodes (EL); providing sensors (PIXEL) associatedwith said electrodes; and displacing said points of stable equilibrium(PEQ) in correspondence with said optical sensors (PIXELS) and/orletting said particles to be displaced in correspondence with saidoptical sensors (PIXELS), wherein to each said electrode (EL) a firstsensor (PIXEL_V) for detecting the transit of said particles in avertical direction, and a second sensor (PIXEL_H) for detecting thetransit of said particles in a horizontal direction are provided. 21.The method of claim 20, comprising providing at least a dichroic filterwith a pre-fixed passband arranged so as to shield at least partly saidsensors (PIXEL), wherein the sensors are optical sensors.
 22. The methodof claim 21, comprising providing a plurality of dichroic filters, eachhaving a passband selected from a set of different passbands, arrangedin a non-superimposed way between them and so as to shield in any caseat least partly said sensors (PIXEL).
 23. The method of claim 21,wherein said at least a dichroic filter is arranged to cover saidelectrodes (EL) and is equipped with interruption openings placed incorrespondence with at least part of each said electrode.
 24. The methodof claim 20, comprising providing at least a dichroic filter with apre-fixed passband arranged in correspondence with a cover (LID)defining together with said array of electrodes (EL) a chamber or amicro-chamber suitable for receiving and containing a fluid samplecontaining said particles.